【作者】 张新萍；

【导师】 杜怡峰；

【作者基本信息】 山东大学 ， 神经病学（专业学位）， 2013， 博士

【摘要】 研究背景Humanin (HN,脑神经肽)是由日本学者Hashimoto在对AD患者枕叶进行DNA功能检测时发现的一个由24个氨基酸残基组成的(Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr-Ser-Glu-Ile-Asp-Leu-Pro-Val-Lys-Arg-Arg-Ala)线性多肽家族,已被体外证明对AD(阿尔兹海默氏病)神经毒性Aβ多肽及β-淀粉样前体蛋白诱导的神经元细胞坏死具有保护作用。Rattin是在鼠类体内发现一种新型的编码38个残基的分泌性脑神经肽类似物(比人类脑神经肽Humanin长14个残基),与人类脑神经肽有73%的一致性,Caricasole A等研究认为Rattin能对抗Aβ淀粉样蛋白诱导的神经细胞毒性及家族性阿尔茨海默病的基因突变,具有抗细胞凋亡作用,且能更有效地对抗NMDA诱导的兴奋性神经元损伤,Rattin的识别鉴定可能是药理学治疗的新途径,其目的旨在提高内源性脑神经肽样类似物的产生。血管性认知功能损害是指由各种脑血管疾病引起的脑功能障碍而产生的一种获得性智能损害综合征,是一种慢性的进行性疾病,已成为继阿尔茨海默病(AD)后的第二大痴呆疾病。混合性痴呆既阿尔茨海默氏病(AD)合并血管性痴呆,临床上并不少见。最近的研究表明,血管性痴呆和AD占据相同的频谱的两端,两者存在共同的血管因素和重叠的临床和病理生理特点。国内外实验证实HN及其衍生物具有潜在的抗细胞凋亡和神经保护活性,拮抗NMDA的神胞毒性作用,且对调节胆碱能系统具有巨大的潜力,对AD,急性缺血性再灌注损伤及药物所致认知功能损伤具有神经保护作用。经文献检索目前HN类似物对慢性脑缺血灌注不良所致血管性认知缺损的研究未见报道,因此我们首次检测HN类似物Rattin在慢性脑血管缺血致认知功能损害大鼠海马和皮层的表达,以证实其对慢性血管性认知损害可能产生的影响。目的通过检测一种新型的HN衍生物Rattin基因及蛋白质在血管性痴呆大鼠脑内的表达,探讨Rattin的表达及其对对认知功能损害可能产生的影响。材料和方法1.动物模型的制备1.1实验动物Animals健康中年雄性SD大鼠30只,体重260-300g,由中国山东中医药大学动物中心提供。随机分为假手术组12只(sham-operated group, SOG),手术组18只(VD model group, MDG)。双侧颈总动脉永久性结扎术(bilateral common carotid artery occlusion, BCCAO)为国际公认血管性痴呆模型常用方法,我们采用BCCAO制备血管性痴呆模型,模拟慢性脑低灌注状态下的认知功能损害。具体操作方法：10%水合氯醛(0.30ml/100g)腹腔注射麻醉,颈前部去毛,消毒后沿腹侧颈正中切开,气管旁分离出双侧颈总动脉,以“0”号线双重结扎颈总动脉。SOG组动物行颈前切开,分离但不结扎颈总动脉。1.2大鼠空间学习记忆能力测试术前1天及术后第31天,采用Morris水迷宫实验测试大鼠的空间学习记忆能力。Morris水迷宫是英国心理学家Morris于20世纪80年代初设计并应用于检测大鼠空间学习记忆的常用装置,分为定位航行实验及空间探索实验两部分,参照有关文献方法于泰山医学院动物实验中心完成测试。1.3行为学测试结果统计学处理：实验数据以x±s表示,组间比较采用t检验,所有数据采用运用SPSS13医学统计学软件处理。应用水迷宫实验对两组大鼠学习记忆成绩进行比较,结果表明MDG组大鼠的逃避潜伏期较SOG组明显延长(P<0.01), MDG组大鼠的跨平台次数较SOG组明显减少(P<0.01)。说明MDG大鼠学习记忆能力降低,造模成功。1.4大鼠标本的取材大鼠模型制作及分组成功,1月取材,用10%水合氯醛0.35ml/100g麻醉大鼠,,常规胸腹联合切口,经左心室插管,分别用生理盐水、4%多聚甲醛的0.1mol/L磷酸盐缓冲液灌注固定,将大鼠断头处死,标记标本的组别和时间点。,随机抽取模型组6只,假手术组6只,从视交叉处向后将大脑切成约3mm厚的薄片,梯度酒精脱水,二甲苯透明,浸蜡,石蜡包埋,以备Nissl染色使用。剩余模型组6只,假手术组6只大鼠迅速分离脑组织,剥取海马,枕叶及颞叶皮层置液氮中,于-80℃低温冰箱保存,分别用于组织总RNA及总蛋白的提取,备real time-qRT-PCR, Westernblotting使用。1.5病理学观察：Nissl染色取鼠海马及颞叶皮层脑切片,依次放入二甲苯和梯度酒精中脱蜡,PBS (0.01mol/L, pH7.4)洗涤后,放入1%的甲苯胺蓝染液中染色数分钟。染色后经95%酒精快速分化,二甲苯透明,最后用中性树脂封片。光学显微镜下观察大鼠海马和颞叶皮层细胞,可见神经元内Nissl体染色呈紫兰色。模型组痴呆大鼠海马CA1区锥体细胞层次减少,排列稀疏,胞浆内尼氏体消失,细胞核体积变小,深染,结构不清,呈核固缩表现,神经纤维排列紊乱；假手术组大鼠海马CA1区锥体细胞2-3层,排列紧密,细胞核圆而大,核仁清晰,胞浆内尼氏体丰富,神经纤维密集,排列整齐；假手术组大鼠颞叶皮层锥体细胞形态正常、结构清晰、Nissl体含量丰富；模型组痴呆大鼠颞叶皮层神经细胞核固缩,局限性神经元数目减少,胶质细胞增生。2.试验方法2.1检测Rattin蛋白在大鼠不同脑区的的表达Western blot分别检测两组大鼠海马,颞叶和枕叶皮层三个部位Rattin蛋白的表达。Rattin蛋白检测：提取Lowry法测蛋白含量。采用Western印迹方法检测Rattin蛋白的相对含量。用凝胶成像分析仪采集图像并用软件进行分析。GAPDH作内参照。操作步骤：(1)组织总蛋白样品的制备(2)细胞总蛋白样品的定量(3)SDS-PAGE凝胶电泳(4)转膜(5)免疫反应(6)膜重新杂交内参GADPH2.2检测Rattin基因在大鼠海马,颞叶和枕叶皮层的表达实时荧光定量PCR分别检测两组大鼠海马,颞叶和枕叶皮层三个部位Rattin基因的表达。RattinmRNA检测方法：用Trizol法提取总RNA; RNA鉴定和定量；反转录反应；PCR反应；取目的基因和GAPDH的PCR产物各5μl,2%琼脂糖凝胶电泳。全自动凝胶成像分析系统照相得到目的基因与GAPDH的扩增产物的积分光密度值(Ⅰ),计算二者的比值(Ⅰ目的基因/IGAPDH),以此作为目的基因的相对表达量。操作步骤：(1)组织总RNA提取(2)反转录反应：RNA反转录成cDNA(3)PCR扩增cDNA:Rattin and β-actin的引物序列：Rattinl5’-TTAGGGACTAGAATGAATGG-3’;Rattin25’-AACTAGTAAATTGAAGCTCC-3’; β-actin a,5’-CCTCTATGCCAACACAGTGC-3’; β-actin b,5’-TCTGCTGGAAGGTGGACAGT-3。配制PCR反应液(反应液配制请在冰上进行)；三步法PCR；融解曲线方法：相对定量；数据处理方法：2-ΔΔC(t)2.3统计学处理：应用SPSS13.0统计软件处理,数据以平均数±标准差(x±s)表示,多组间均值的比较用SNK法单因素方差分析。结果1.模型组(MDG)与假手术组(SOG)Rattin蛋白表达量比较MDG组大鼠颞叶及枕叶皮层Rattin蛋白表达量与对照SOG组比较未见明显差异(P>0.05,P>0.05); MDG组大鼠海马与颞叶及枕叶皮层Rattin蛋白表达量对比明显增高(P<0.01,P<0.01); SOG组大鼠海马与颞叶及枕叶皮层之间Rattin蛋白表达量比较明显降低P<0.01,P<0.01), SOG组大鼠颞叶及枕叶皮层之间Rattin蛋白表达量比较未见明显差异(P>0.05)。2.模型组与假手术组Rattin基因表达量比较模型组(MDG)与假手术组(SOG)之比结果分析采用2-ΔC(t)方法。在基因水平上对Rattin进行定量分析,通过荧光实时PCR检测显示：MDG组与对照组颞叶和枕叶Rattin表达比较无显著差异(P>0.05,P>0.05); MDG组海马区Rattin基因表达量比对照组明显增高(P<0.01)；SOG组海马与颞叶,枕叶比较Rattin表达有显著降低(P<0.01, P<0.01), SOG组颞叶,枕叶间Rattin表达无明显差异(P>0.05)。讨论近几年的研究发现HN类似物Rattin对AD及AD以外的认知功能缺损有神经保护作用,其机制可能与抗NMDA毒性,调节神经胆碱乙酰转移酶和囊泡内乙酰胆碱运载体mRNA表达有关,同时具有调节氧化应激反应,减少细胞凋亡,保护血管内皮细胞的功能,HN类似物的分泌活性是在其基因序列中进行编码的,HN必须被分泌到细胞外才能发挥它的抗AD相关损伤的神经保护功能。目前认为人类HN多表达在能量代谢相关的组织,如心脏,骨骼肌,肾脏和肝脏,在免疫系统中几乎未检测到,在脑区小脑和枕叶比其他部位表达更显著,脑脊髓液,精液和血浆中也见表达。Yun K. Oha发现在人类乳房内动脉,动脉粥样硬化的冠状动脉,大隐静脉的内皮细胞也可见HN的表达,HN可通过调节进展斑块的氧化应激反应和细胞凋亡保护血管内皮细胞功能,防止动脉粥样硬化斑块的进展。Caricasole A报道了鼠类HN类似物基因,命名为rattin (RN),证明Rattin是一种新型种族发育相关性脑神经肽,具有针对Aβ和兴奋性神经元死亡的保护活性。我们首次在慢性脑灌注不良所致认知功能损害的大鼠模型海马,枕叶,颞叶中检测Rattin的表达,以探讨其可能发生的改变。本实验通过Western blot, RT-PCR方法检测HN类似物Rattin蛋白及基因在血管性痴呆大鼠海马和颞叶,枕叶皮层的表达,首次发现慢性脑灌注不良致血管性痴呆大鼠海马Rattin表达显著增高,而颞叶,枕叶皮层Rattin表达没有变化,此结果提示海马神经细胞可能通过自身分泌Rattin发挥一定的神经保护作用,Rattin表达增高可能与学习记忆损害有关,这种增高可能是一种反应性神经保护作用。海马神经元缺血可致神经元皱缩,数目减少,细胞内胞质贫乏,核糖体、线粒体和内质网数量减少,胞浆内含有大量溶酶体和细胞丝,突触前膜或后膜肿胀、空化,由此造成与认知损害有关的神经递质如乙酰胆碱,兴奋性氨基酸,神经肽等异常,同时激发Rattin的表达增高。Terashita K,研究显示人脑中脑神经肽在小脑和枕叶的表达高于其他脑区,正常大鼠各脑区Rattin在海马的表达最低,枕叶最高,本实验大鼠正常对照组海马,颞叶和枕叶Rattin表达未见显著差异,另一方面,双侧颈动脉永久性结扎致慢性脑缺血累及海马,颞叶为主,颞叶皮层受损亦可引起近事记忆缺损,实验显示颞叶Rattin表达没有变化,此结果及机制有待于进一步研究。结论Rattin作为一种新型脑神经肽类似物对慢性缺血性血管性痴呆可能具有一定的神经保护活性,此结果为血管性认知缺损发病机制的研究和药物治疗提供新的方向。更多还原

【Abstract】 BackgroundHumanin (HN) is a newly discovered24-amino acid peptide, which may suppress neuronal cell death. HN cDNA includes an open reading frame (HN-ORF) of75bases located950bases downstream of the5’end of theHNcDNA. It has been demonstrated thatHN cDNA is99%identical to the mitochondrial DNA (mtDNA) sequence. HN homologs have been identified as expressed sequence tags (ESTs) in both rats and nematodes. Certain regions that are homologous to the HN cDNA exist on human chromosomes. HN forms homodimers and multimers and this action seems to be essential for peptide function. HN acts as a ligand for formyl peptide receptor-like1(FPRL1) and2(FPRL2). It has been demonstrated that HN plays a protective role through its antiapoptotic activity that interferes with Bax activation, which suppresses Bax-dependent apoptosis. HN has also been shown to suppress the c-Jun Nterminal kinase (JNK) and ASK/JNK-mediated neuronal cell death. Several studies have also confirmed that HN could be important in the prevention of angiopathy-associated Alzheimer’s disease dementia, diseases related to mitochondrial dysfunction (MELAS), and other types of amyloid accumulation-associated neurodegeneration. A very recent study demonstrated a pluripotent cytoprotective effect and mechanisms of HNs in cells not from the CNS, such as germ cells or pancreatic cells, and the potent physiological consequences that result from HN interaction with IGFBP3and STAT3. In vivo studies suggest that HN may also protect against cognitive impairment due to ischemia/reperfusion injury.In summary, the cytoprotective effects of HNs seem to be associated with antiapoptotic, metabolic (improving mitochondrial bioactivity) and possibly anti-inflammatory effects by the way of competition with hormone carrier proteins and cellular receptor agonists. To unravel the pluripotential mechanism(s) of action, bioavailability and regulation of expression and the fate of endogenous HN peptide(s), further studies are needed.In vivo studies, of which there have been relatively few, show that HN may protect against cognitive impairment, inflammatory response, apoptosis and stroke induced by different factors such as scopolamine,3-quinuclidinyl benzilate or brain ischemia caused by the middle cerebral artery occlusion in mouse or rat models.Very recent studies revealed that HN can also play an important role in the regulation of glucose homeostasis.The identification of a novel rat cDNA encoding a peptide homologous to Humanin was reported, a secreted peptide that specifically protects against neuronal cell death induced by beta-amyloid peptide (A β) or by mutations causing early-onset familial Alzheimer.s disease. The rat gene, which we termed Rattin, encodes a peptide of38residues (14residues longer than Humanin) showing73%identity in the conserved region to Humanin. The expression profile of the1.6-kb Rattin transcript is comparable to that displayed by Humanin, with significant expression levels in the central nervous system and in cardiac and skeletal muscle. The full-length Rattin peptide and its1-25fragment were equally effective as Humanin in protecting rat-and mouse-cultured cortical neurons against Aβ-induced toxicity. However, Rattin was much more effective than Humanin against excitotoxic neuronal death induced by a toxic pulse with NMD A. Rattin and its short fragment were protective against excitotoxic death not only when coapplied with NMDA, but also when added to the cultures after the NMDA pulse. Neither Rattin not Humanin could affect neuronal apoptosis by trophic deprivation induced in cultured cerebellar granule cells depleted of extracellular potassium. This suggests that Rattin is the prototype of a novel class of peptides, phylogenetically related to Humanin, endowed with protective activity not only against A β but also toward excitotoxic neuronal death. The identification of Rattin may be instrumental for the development of novel pharmacological strategies aimed at enhancing the production of endogenous Humanin-like peptides.The availability of Rattin should facilitate studies aimed at elucidating the mechanism of action of Humanin-like peptides and the study of their pharmacological properties in vivo. Vascular dementia (VaD) is a clinical syndrome of cognitive decline caused by cerebral vascular diseases. It is the second most common type of dementia after Alzheimer disease (AD). In China, it accounts for approximately45.8%of dementia cases and has an increasing impact on an aging population. Chronic cerebral hypoperfusion is the main mechanism for VaD, and permanent bilateral common carotid artery occlusion (BCCAO) in rats is a suitable model for the studies of VaD. In the present work we investigated the changes of Rattin expression in different region such as hippocampus, occipital and temporal lobes of the rat model of vascular dementia.PurposeTo investigate the effects of Rattin expression in the hippocampus and cerebral cortex of the rats model with vascular dementia (VD) induced by chronic cerebral hypoperfusion,Materials and Methods1.AnimalsSprague-Dawley male rats (weighing260-300g) were obtained from Animal Center of Shandong University of Traditional Chinese Medicine. Rats were housed under conditions of controlled lighting (12:12h light-dark cycle) and temperature (25±2℃), and had free access to food and water ad libitum. All procedures were performed in accordance to the Guidelines of the Animal Care Committee of Shandong University.2.Surgery ProcedureThe animals were anesthetized with chloral hydrate and carried out BCCAO. During the surgery body temperature of rats was continuously monitored with a rectal probe maintaining at37±0.5℃. Both common carotid arteries were carefully exposed and separated from the vagus nerve which was permanently ligated with a5-0silk suture. The sham-group carried the same surgeries without ligating the bilateral common carotid arteries. After surgeries rats were kept in cages to recover from anesthesia.3.Behavioral Measurement One month after surgeries, the rats were performed by Morris water maze to evaluate the learning and memory function as described before [13]. The Morris water maze consisted of a pool (1.6m diameter) and filled with water (23±2℃, depth35cm). The pool was divided into four quadrants. The platform was submerged about1.5cm below the water surface and placed at the midpoint of a fixed quadrant. All performance of the rats in the pool was recorded by a video camera (MI-200, Chengdu Taimeng Technology&Market Co., Ltd., China). Memory-acquisition trials (training) were performed four times daily for6days and escape latencies (time of finding submerged platform) were measured. The rats were trained for6consecutive days with4trials per day (an interval of20min) to find the platform. They were allowed to find the platform in120s and left on the platform for30s. If the rats couldn’t find the platform within120s, they were directed toward the platform by the observer and placed on the platform for30s. On the7th day, the platform was removed and the rats were allowed to swim in the pool for120s. The number of crossings over a point where the platform had been was recorded.4.Nissl stainingThe brains were taken out and cut after4%paraformadehyde perfusion. The sections were selected with Nissl staining. Then the brain sections were observed with microscope and taken photos.5.Western BlotAfter the behavioral test, the rats were decapitated under anesthesia. Brains were immediately taken out, and hippocampus, occipital and temporal lobes were isolated for Western Blotting detecting. All the tissues were lysed in lysis buffer and the protein content of the supernatant was measured. Equivalent amounts of protein from each sample were separated using10%SDS polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to PVDF membranes in a Tris-glycine transfer buffer for1h at4℃. The membranes were washed and blocked in5%non-fat dried milk overnight at4℃using primary antibodies against Rattin (Novus) and GAPDH (Santa Cruz). At2nd day, the membranes were taken out for1h at room temperature and incubated with an appropriate horseradish peroxidase-conjugated secondary antibody. Rattin expression was detected by ECL kit.6.Real-time PCRAfter the rats were sacrificed by anesthesia, the tissues were taken and homogenized, hippocampus, occipital and temporal lobes were isolated for Real-time PCR. Total RNA was extracted by Trizol reagent. The primer sequences for Rattin and β-actin used were as:follows:Rattinl,5’-TTAGGGACTAGAATGAATGG-3’; Rattin2,5’-AACTAGTAAATTGAAGCTCC-3’; β-actin a,5’-CCTCTATGCCAACACAGTGC-3’; β-actin b,5’-TCTGCTGGAAGGTGGACAGT-3’ The results were expressed as the ration of BCCAO/control by using2-ΔΔC(t) method.7.Statistical analysisAll the data were expressed by mean±S.E.M.. The differences between groups were analysed by ANOVA followed by Student-Newman-Keuls test. P<0.05was considered as significance. Results1.Morris water maze performanceMorris water maze showed that the behavioral function was damaged in the rats with BCCAO. The time of escape latency increased and the number of crossing the place of the platform decreased significantly compared the control group (n=6, P<0.01)2.Morphological changes of neurons in cortex of hippocampus and temporal lobeThe morphology of neurons in hippocampus and temporal lobe of BCCAO rats was damaged. Number of neurons markedly decreased, dissolution of Nissl’s substance and pyknosis of the neuclei compared with control group. The similar changes were present in temporal lobe of BCCAO ratscompared with control group.3.Changes of Rattin protein expression in hippocampus, occipital lobe and temporal lobeRattin expression in hippocampus of the rats with BCCAO increased significantly compared the control group (n=6, P<0.01). However, Rattin expression in occipital and temporal lobes of the rats with BCCAO did not changed significantly compared the control group (n=6, P>0.05). In addition, Rattin expression in hippocampus was lower than occipital and temporal lobes in control group.4.Changes of Rattin mRNA expression in hippocampus, occipital lobe and temporal lobeIn rat model of vascular dementia, Rattin mRNA expression in different regions of brain were measured using real-time PCR. The primers of Rattin and β-actin worked well. Different transcriptional effects of rattin gene in different brain zone response to BCCAO. The mRNA content changes of rattin were detected using by real-time PCR, β-actin as inner. Rattin mRNA expression increased significantly in hippocampus (n=6,*P<0.01), but not changed obviously in temporal and occipital lobes, compared to individually control group (Hippocampus, temporal and occipital) DiscussionPermanent BCCAO in rats has been widely used as a model to study VaD. In the rat BCCAO model, BCCAO causes cerebral blood flow to decreass immediately after occlusion and is an interesting approach to evaluate the effects of cerebral hypoperfusion. This leads to contributes to behavioral and cognitive deficits. Chronic cerebral hypoperfusion results in neural damage to the hippocampus, the cerebral cortex, the white matter areas and the visual system. The hippocampus is the most important position which is associated with learning, memory and other cognitive functions, and Chronic cerebral hypoperfusion first involved the hippocampus.The expression of Rattin in the adult rat shows a ubiquitous transcript distribution with marked tissue-specific differences in mRNA levels. In central nervous system, Rattin transcript exists in hippocampus with lower levels than cortex and cerebellum. It is also reported that Rattin is neuroprotective against A|3-induced neurotoxicity. In the present work we first measured the expression of Rattin protein in hippocampus, occipital and temporal lobes of rats with BCCAO. In hippocampus the level of Rattin protein increased significantly, but the levels of Rattin protein did not change in occipital and temporal lobes. This might be related to the pathological changes of VaD. Rattin protein in hippocampus was lower than occipital and temporal lobes in control group, which is parallel with Rattin transcript (mRNA) exists in hippocampus. In VaD rat model, the expression of Rattin increased, which might be related to the neuroprotection. The high expression levels of Rattin protein in hippocampus might be associated with vascular cognitive impairment, and might be a neuroprotective response. On the other hand, the levels of Rattin protein did not change in occipital and temporal lobes, which might be related to the other pathological changes of chronic cerebral hypoperfusion.ConclusionThe high expression levels of Rattin protein in hippocampus might be associated with vascular cognitive impairment, and might be a neuroprotective response. The results will contribute to the studies of mechanisms for VaD and provide the potential therapy in future.更多还原